Deciphering waste bound nitrogen by employing psychrophillic Aporrectodea caliginosa and priming of coprolites by associated heterotrophic nitrifiers under high altitude Himalayas

Himalayan ecosystem is characterized by its fragile climate with rich repositories of biodiversity. Waste collection and disposal are becoming increasingly difficult due to topographical variations. Aporrectodea caligenosa, a versatile psychrophillic soil dweller, is a useful biocatalyst with potent bio-augmented capability for waste treatment at low temperatures. Microcosm experiments were conducted to elucidate the comprehensive nature of biogenic nitrogen transformation to NH4+ and NO3− produced by coupling of earthworm-microbes. Higher biogenic recovery of NH4+-N from coprolites of garden soil (47.73 ± 1.16%) and Himalayan goat manure (86.32 ± 0.92%) with an increment of 14.12 and 47.21% respectively over their respective control (without earthworms) with a linear decline beyond 4th week of incubation was reported. NO3–-N recovery progressively sustained in garden soil and goat manure coprolites during entire incubation with highest 81.81 ± 0.45 and 87.20 ± 1.08 µg-N g−1dry weight recorded in 6th and 5th week of incubation respectively and peak increments as 38.58 and 53.71% relative to respective control (without earthworms). Declined NH4+–N in coprolites at low temperature (15.0 ± 2.0 °C) evidenced increased nitrification rates by taking over the process by abundant nitrifying microbes. Steady de-nitrification with progressive incubation on an average was 16.95 ± 0.46 ng-N g−1 per week and 21.08 ± 0.87 ng-N g−1 per week compared to 14.03 ± 0.58 ng-N g−1 per week and 4.50 ± 0.31 ng-N g−1 per week in respective control treatments. Simultaneous heterotrophic nitrification and aerobic denitrification (SHNAD) was found to be a prominent bioprocess at low temperature that resulted in high and stable total nitrogen and nitrate accumulation from garden soil and goat manure with relative recovery efficiency of 11.12%, 14.97% and 14.20%; 19.34%. A. caligenosa shows promising prospects for mass applicability in biogenic N removal from manure of Himalayan goat.

, Mg, P and K relative to surrounding soil 13,28,29 . The nitrogen transformation is an important component of waste conversion during composting and vermicomposting processes [30][31][32] and processes like nitrification and denitrification are the key activities contributing to nitrogen cycle, playing an important role in nutrient supply, (NH 4 + -N), volatilization and greenhouse gas (GHG) emissions for various ecosystems 30,33 . Most of the reported heterotrophic nitrifying-aerobic bacteria are mesophilic bacteria, carrying out nitrification and denitrification at temperatures ranging from 15 to 37 °C 34,35 . Temperate microbes have obvious physiological advantages 36 and unique ability to perform nitrification and denitrification synchronously 37 and improve the overall nitrogen removing efficacy at lower temperatures. Guraz valley, a fragile cold ecosystem, located in high Himalayas with prominent features of hill agriculture and diverse habitats rich in biodiversity. The disparate topographic features offer microhabitat for a variety of organic herbal crop species to grow in the main cultivated area while woody Betula pendula, Pinus roxburghii, Quercus robur and Cedrus deodara grow in the surrounding forests 38 . All the three ethnic Himalayan communities (nomadic Bakerwals, seminomadic Gujjars and semi-sedentary Kashmiri shepherds) depend on natural resources 38 . Since, there is very insufficient and scanty information regarding wastes recycling and recovery of nitrogen nutrient in the Himalayan ecosystem. Nitrogen recovery from waste material has now become a pivotal issue globally (Wang et al. 2015; Chen et al. 2016). Open dumping of bio-wastes is considered a potent source of nitrogen pollution in different forms viz; NH 3 and N 2 O to air and N-NO 3 − to ground and surface water resources 39,40 . Changes in transformation/emission rates are affected by the soil temperature, because it affects the activity of urease, nitrifier communities, and nitrification rate in the soil. Transition in NH 4 + -N, NO 3 − -N and total nitrogen in the interim of vermicomposting had been reported, however, divergence and succession of N-transformation process through involving functional psychrophillic microbes and earthworms were seldom reported. Therefore, the information on cold tolerant earthworms and associated nitrifying and denitrifying microbial species can furnish novel insights into the N transformation during the vermicomposting process in cold habitats. Moreover, the positive priming through the endogeic geophagous earthworm's influence is expected to foster the recycling of nutrients, especially organic carbon, nitrogen and phosphorus 41,42 . The present study, which is perhaps the first of its kind under cold habitat of Guraz valley in Kashmir region, will pledge cognition and contribute to sound understanding of mineral N dynamics using psychrophillic A. caligenosa indigenous to the Guraz valley, with the goal as (i) o determine the involvement of coprolite associated microorganisms in bioconversion of N, (ii) to enumerate the changes in N-NH 4 + , N-NO 3 − and (iii) specifically, recycle and evaluate the impact of locally available garden soil and Himalayan goat manure on physico-chemical flux's in coprolites resulting in minimizing nitrogen pollution in temperate ecosystems.

Results and discussion
Ammonium and nitrate dynamics. The analysis performed on coprolites of Aporrectodea caliginosa evidenced that ammonium (NH 4 + -N) concentration after one week of incubation were significantly affected by the source of food (Fig. 1). As evident from Fig. 1 the concentration of NH 4 + -N in all the earthworm released coprolites increased steadily upto the 4th week of the vermicomposting process, however, coprolites of goat manure (GM) shows continuous increase in NH 4 + -N content upto the 5th week. The NH 4 + -N concentration in GM coprolites was significantly (p < 0.05) higher than GM (control) concentration upto 5th week of incubation, after which the NH 4 + -N concentration began to fall steadily until the 6th week of incubation. Further, all treatments showed decline in the NH 4 + -N concentration after 4th week continued upto the 6th week of incubation. It was observed that after 6 weeks of vermicomposting the NH 4 + -N concentration in the coprolites was in the order; GM (control) ( (Fig. 1). Earlier research also suggests that ammonification is a temperature sensitive microbial mediated mineralization process [46][47][48] and an increase in temperature upto 25 °C will significantly increase the transformation rate of N 49 . The trend for nitrate (NO 3 − N) concentration varied between 8.37 and 81.88 µg NO 3 -N g -1 DW in coprolites influenced by different treatments. The NO 3 --N concentration of coprolites from earthworms fed on GM and GS was found higher. NO 3 --N concentration showed a linear increase from the 1st to 6th week of vermicomposting from coprolites of both GM and GS with the highest values recorded as 90.2 µg NO 3 -N g -1 DW and 81.81 µg NO 3 -N g -1 DW respectively. The usefulness of earthworm ' s gut microbes inflates the process of nitrification compared to the situation without earthworms 43,50 . The NO 3 --N removal from the GM was exceptionally high and ranged between 22.03 to 90.20 µg-NO 3 g -1 DW with an average value of 64.24 µg-NO 3 g -1 DW during the entire incubation period (Fig. 2). Similarly, for GS NO 3 --N recovery ranges from 8.37 to 81.81 µgNO 3 g −1 DW. Further, our findings reveal a linear increase in NO 3 --N concentration from GM coprolites along the dura-  www.nature.com/scientificreports/ tion of incubation, which is assumed to be correlated with a higher concentration of NH 4 + -N in coprolites, which is a primary source for the nitrification process. Our results also suggest that continuous escalation in NO 3 --N could be due to the involvement of heterotrophic bacteria and fungi in coprolites (Table 1) preferring a moderate temperature between 15 and 25 °C which also shows that heterotrophic nitrification process exceeds autotrophic nitrification. Previous research findings suggest that earthworm gut and castings tend to have more active heterotrophic microbes that positively influence the amounts of extractable NO 3 --N in upper layer of soil 51-53 moreover, increased NO 3 − concentration in coprolites is a good indicator of the nitrification process and abundance of nitrifiers 23,25,[54][55][56] . It is also reported that the heterotrophic nitrification has an optimum temperature requirement of 15 °C 57 while, it exceeds over autotrophic nitrification if the temperature is increased to 35 °C 49 . Further, we confirm that the heterotrophic nitrification is an ecosystem dependent process which is directly dependent on substrate type, microbial diversity and abundance. The samples of study material yielded a large number of indigenous heterotrophic nitrifiers, which were determined to have high nitrogen removal abilities. Temperature stimuli, NH 4 + -N and NO3 --N content in substrates influenced the overall performance of heterotrophic nitrification process. Our previous research in the cold arid Ladakh and Kashmir valley backs up this argument. The influence of low temperature on heterotrophic nitrification at the experimental site (Guraz valley) is consistent with the previous findings, which demonstrated that an optimal temperature, substrate type and ecology conditions favor heterotrophic nitrification process 13,58,59 . Low concentration of NO 3 − N is related to low microbial dominance (nitrifying bacteria) in coprolites from control treatments of both the substrates suggesting the loss of potency in coprolites which inhibited the nitrification process. Lower functional redundancy in the nitrification process is due to the lack of earthworms in the control treatments which could not either enhance biogeochemical stability of organic matter or stimulate microbial activity (Table 1). Thus, the results clearly elucidated an amicable relationship between earthworm and heterotrophs which favors the heterotrophic nitrification. Previous studies support the increased mineralization of nutrients in earthworm coprolites relative to surrounding garden soil (without earthworm), which is associated with enrichment in liable compounds due to various factors such as, increased activity of nitrifying microbes 60 , digestion of earthworm could influence gut microbiome 61 and earthworm-microbe association produces enzymes that are reported to increase NO 3 − N and NH 4 + -N content in coprolites by 31 and 14% respectively 62-65 . Denitrification/ N 2 O emission during incubation. The interaction between earthworms and denitrifers directly affect the nitrogen dynamics via nitrous oxide (N 2 O) fluxes. Our study reveals that denitrification predominantly turned out to be limited by low temperature and C supply. N 2 O emission rates from mesocosm surface coprolites (soil fed earthworms) ranged from 5.90 ± 0.20 ng-N g −1 (1st week) to 16.6 ± 0.48 ng-N g −1 (6th week) with an average of 16.95 ng-N g −1 (Fig. 3). However, the emission rates of N 2 O from the control treatments, on the hand were relatively low ranging from 4.05 ± 0.29 ng-N g −1 (1st week) to 14.60 ± 0.22 n g-N g −1 (6th week) with an average of 14.0 ng-N g −1 per week. N 2 O emissions showed a steady increase along with the incubation period except from the 6th week onwards, there was a downward trend, with an average increase of 17.03% in emission from coprolites of GS compared to GS (control) . Earlier research has shown that A. caliginous is capable to emit significant amounts of N 2 O emissions from the soil through different activities [66][67][68] . Earthworms have the potential to dramatically regulate the physico-chemical properties of their habitats and thereupon affecting the production of GHG 20 however, denitrification is affected by a variety of environmental factors including availability of dissolved oxygen (DO), carbon (C), pH, temperature, denitrifying bacterial population and congregations of NO 3 − , NO 2− and S 2−31,69,70 . www.nature.com/scientificreports/  www.nature.com/scientificreports/ Under mesocosm condition, incubation of earthworm with GM stimulates the denitrification process, which showed a favorable relationship with N 2 O emission from manure coprolites ranged from 7.26 ± 1.32 ng-N g −1 (1st week) to 20.19 ± 1.03 ng-N g −1 (6th week) (Fig. 4). On an average N 2 O emission from coprolites of GM was 78.65% higher compared to GM (control) , suggesting that A. caliginosa is playing a critical role in conversion of excess NO 3 − to N 2 O. It might be attributed to the higher initial availability of carbon in goat manure (Fig. 5), which constituted 70% of the total mass in this substrate, led to faster degradation and enhanced mineralization of N to NO 3 − . Limited reports are available about the mechanism involved in the combined relationship between the nitrifying and denitrifying microbes that help towards better understanding of the N-cycle in terrestrial ecosystem 71,72 . Bioconversion of cow dung, duck manure, kitchen waste by earthworms is reported to induce N 2 O emissions 54,73 ; however, emission rates are significantly lower than thermophiles composting 74,75 . Vermicomposting under high moisture conditions were reported to decrease N 2 O and CH 4 emissions by 25-36 and 22-26% compared to thermal composting 74 . Increased N 2 O emission in GM coprolite must be owing to the activities of denitrifying bacteria, as evidenced by significant (p < 0.05) difference in N 2 O emission between samples from GM and GM (control) treatments. The research also found a link between nitrification and denitrification (Figs. 4, 8), and demonstrate how a heterogeneous microbial population and function may coexist. Previous research also found that potential denitrification rates were positively correlated with coexistence of aerobic and anaerobic microbes (denitrifers) [76][77][78][79][80] . In our study, low N 2 O accumulation was found in headspace of bottles with no C 2 H 2 or with low concentration for both control treatments of GS (control) and GM (control) . However, significant (p < 0.05) difference in N 2 O emission was evidenced between the two control treatments GS (control) and GM (control). On an average, N 2 O produced from the coprolites fed on GM were 19.57% higher compared to castings obtained from GS.
Physico-chemical analysis of worm casts. Coprolites produced from GS and GM were rich in nutrients and A. caliginous had a substantial impact on the analyzed parameters. Significantly (p < 0.05) higher pH was observed in coprolites from GS and GM treatments relative to the GS (control) and GM (control) (Fig. 5, 6). The final pH values of both the coprolites from GS and GM were slightly acidic to alkaline attributed to gut microbial activity, indicating that these coprolites could be useful to remediate soil reaction. Vermicomposting of fruit and vegetable wastes 81,82 ; seaweeds, sugarcane trash, coir pith amended with cow dung 83 ; and flowering plants (Lantana camara) 84 was also reported to produce vermicompost with a pH close to neutral. Analyses of variance for total organic carbon indicated significant (p < 0.05) differences between the coprolites from GS (18.24%) and GM (27.18%) with their controls GS (control) (29.42%) and GM (control) (32.02%) respectively. A linear decline in total organic carbon from GS and GM coprolites is attributable to utilization of carbon by microbes during the entire process (Fig. 7). Previous reports have also mentioned the loss of 19-67% of carbon during the process of vermicomposting 85,86 where dehydrogenase activity plays a key role in the hydrolysis of cellobiose and other 6  www.nature.com/scientificreports/ disaccharides during vermicomposting process 82 . It has been also reported that the chief mechanism for the carbon loss from the substrates could be attributed to the respiration of earthworms and microorganisms during the decomposition and transformation of substrates 87,88 . The Fig. 7 depicts a significant (p < 0.05) increase in NO 3 − by 15 and 39% in coprolites of GS and GM respectively, when compared to GS (control) and GM (control) . Increased NO 3 content in coprolite is attributed to the significant influence of gut associated nitrifying microbes in the production of NH 4 + which is a primary substrate for NO 3 − yield, in addition, earthworm mucus and nitrogenrich excretory secretions also contributed to NO 3 − content. The NH 4 + concentration is also correlated with the initial N content of waste substrates, which was 1.85 ± 0.04 and 2.14 ± 0.05% in GS (control) and GM (control) , respectively (Fig. 7). Previous research have noted that earthworms may ameliorate the castings as a consequence of N transformation from wastes by associated microbes through bio-waste mineralization and gut N-fixation 21,[89][90][91] . Total N concentration in the coprolites significantly (p < 0.05) increased and could be interpreted due to the factors such as: initial N content of the substrate; bioconversion efficiency; possible death of a few baby worms; secretions of mucus, addition of nitrogenous substances during the entire process. Gut and skin of earthworm can secrete nitrogenous compounds which is also one of the reasons for enriched N content in the end product of the process 86,92 . At the end of vermicomposting process, the total phosphorus (TP) recovery was found 6.15% in GM and 8.19% GS from the respective substrates (Fig. 7). Increased P concentration in the GM castings could be due to secretion of various organic acids by related microbes and decomposition of substrates by earthworm, as corroborated with previous studies 93, 94 . Table 1 shows that the bacterial population in both coprolites from GS and GM increased that includes Aerobacter sp., Bacillus sp., Citrobacter sp., Escherichia sp., Klebsiella sp., Pseudomonas sp., Proteus sp., Serratia sp. and Staphylococcus sp. The coprolites of GS and GM demonstrated significantly (p < 0.05) higher bacterial and fungal density compared to respective controls GS (control) and GM (control) .
The earthworm amended treatments demonstrated significantly (p < 0.05) higher bacterial density than GM (control) . Similarly, higher fungal density was also observed in earthworm inoculated coprolites of GS and GM. The fungal species included Aspergillus sp., Fusarium sp., Penicillium sp. and Saccharomyces sp. The presence of more bacteria and fungi in earthworm amended coprolites indicate that earthworms could favor and compliment the microbial communities during conversion of substrates. This is in accordance with earlier results of using mill waste 95,96 , forest litter waste 97 , sewage sludge and rice straw 31 as substrates in vermicomposting which favors the microbial population. It has also been reported that presence of the earthworms in vermicomposting enhances the beneficial microflora and suppresses harmful pathogenic microbes 98 , that later enhance plant growth via the production of plant growth promoting compounds. Thus, the higher population of microbes in the earthworm castings could be due to the abundance of beneficial microbes in the earthworm gut. This concept was also supported by the findings of previous research 99, 100 . The potential availability of nutrients in coprolites 8 Figure 6. Physico-chemical characteristics of garden soil (initial) and coprolites of garden soil (GS) with and without A. caliginosa (mean ± SD). Corresponding means followed with same letters are not significantly different at P < 0.05. www.nature.com/scientificreports/ was revealed by regression analysis between the indices of physico-chemical properties of GM ( Table 2). The Fig. 8 shows that the selected parameters were appropriate to determine the stabilization of coprolites in the current investigation.   www.nature.com/scientificreports/ Nitrification potential. Replicated sampling analysis at intervals revealed an increase in potential nitrification rates, demonstrating a continuous nitrification process in coprolites in all the treatments except control (Fig. 9). The nitrification sampling in triplicates was done at three different stages of vermicomposting; nitrification-I (2nd week), nitrification-II (4th week) and nitrification-III (6th week) to estimate NO 3 − levels. Nitrification rates increased over ammonification, as performed by the involvement of both nitrifying bacteria and fungi ( Table 1). The mean potential nitrification rates of GS and GM coprolites at three stages of the nitrification process were 4.63, 43.46, 51.21 µN g −1 DW and 6.48, 130.43 135.71 µN g −1 DW respectively that were significantly (p < 0.05) different from the potential nitrification rates of the respective GS (control) & GM (control) (Fig. 9). Abundances of nitrifying bacteria and nitrifying fungi increased nitrification during the entire incubation period, apart from that nitrification potential by earthworm, it also depends on the initial N content of organic substances used by earthworms as a source of food. The study evidenced that unlike other microbial processes, nitrification progressed with a steady increase in NO 3 − concentration at all the three stages of nitrification process. In contrast, the net rate of nitrification was comparatively low in the GS (control) & GM (control) . Previous studies indicate that earthworm castings have strong associated activity for nitrification [101][102][103] . Our findings,  www.nature.com/scientificreports/ support the hypothesis that earthworm activities with consumption of N-rich food material could increase the nitrification process in coprolites which is also supported by earlier research 44,91,104 .   Experiment. The present study used A. caliginosa, which is found in the garden and coniferous forest soils of Guraz valley 105 . Garden soil (GS) from a field planted with maize + beans and goat manure (GM) from Bakerwal (Himalayan shepherds with rearing Himalayan goats) were used as substrate treatments in triplicates with corresponding control as designated in Table 4. Earthworms were placed in 12-mesocosms (polyvinylchloride-PVC) with a diameter of 550 cm 3 filled with substrates separately (Fig. 11). The activity of nitrogen dynamics from the earthworm coprolites was sampled three times in a week. Mesocosms were filled with the same feeding material and inoculated with ten (10) non-clitellated young worms for 42-45 days. Surface coprolites samples were shifted to petri dishes with moistened filter paper for further analysis. The mesocosms were placed under ambient light, with an average air temperature and relative humidity of 16.8 ± 1.5 °C and 67 ± 4% respectively, to ensure similar microclimatic conditions during the course of the experiment. ) solution. Foam plugged flasks were shaken mechanically at 25 °C for 2 h. A mixture sample of 10 ml was taken for N 2 O analysis. Flasks were then exposed to aerobic conditions for two days followed by filtration and the amount of N 2 O was measured. The difference between the initial and final readings of N 2 O was recorded as the nitrification potential. Nitrification rates were expressed on a dry weight basis. During the experiment, denitrification rates were determined three time (2nd, 4th and 6th weeks) using surface coprolites taken from each mesocosm. All castings were removed from mesocosm surface two days before coprolite sampling, so all coprolite samples evaluated for denitrification in the experiment were ≤ 2 days old. The acetylene block method 108 was used to determine denitrification rate as: Coprolite sample (2 g fresh weight) of A. caliginosa from each mesocosm surface were placed in10 ml test tubes fixed with headspace, and 1.0 ml of hydrogen and carbon was added to the headspace of each test tube to inhibit the reduction of N 2 O to N. N 2 O production was measured every 3 h for 14 h at 16 ± 2.5 °C. Modified automated method 109 , was used to determine the N 2 O.

Measurements. Surface coprolites of
Coprolite microbiome. For differential analysis of the micro flora, coprolites samples were sub-sampled.
In addition, earthworms were put under starvation for two days in petri dishes with 1% sterile agar. This much time was sufficient to get the transit microbes come over the agar. Coprolite samples were dissolved into 10 ml of sterile 0.85% NaCl and stirred vigorously for 20 min using the method 110 described as: Suspension was diluted by the serial dilution method using a dilution factor of 10 -1 -10 -10 to make out the development of microbes on agar nutrient plates. Enumeration of microorganism was carried out by pour plate method on nutrient agar. Microbial samples were incubated for 24 h at 20 ± 2 °C. Using the purified streak-plate technique, each isolate with similar morphological features was eventually relocated to a new nutrient agar plate until a single colony was established. Pure microbial colonies were characterized and identified by perceiving morphological features and bacterial cell shapes through the gram staining technique 111 . Nitrogen transformation activity of pure isolates were tested by using Kjeldahl method 112 , on the basis of generation of initial turbidity in flasks with nitrogen free medium.
Chemical analysis. Coprolite samples were ignited in Muffle furnace at 500 °C for 90 min for determination of organic carbon using the method as described by Nelson and Sommer (1996). Phosphorus and potas- www.nature.com/scientificreports/ sium were quantified by the procedure of John (1970) through flame photometer-128 (Systronics) after digesting samples in a diacid suspension (HClO 4 : HNO 3 in the ratio of 4:1). pH and electric conductivity (EC) were determined in double-distilled water blend each with a concentration ratio of 1:10 (w/v) plying digital meter (COM-100) and Eqip-tronics (EQ-614A) respectively. Nitrogen (N) was determined by the Micro-Kjeldhal method as described by Bremner and Mulvancy (1982) using digestion extract (H 2 SO 4 + K 2 SO 4 : CuSO 4 : SeO 2 in the ratio of 10:4:1). Phosphorus (P) content was determined by nitro-vanadomolybdate method, potassium (K) by using photometry and micronutrients (Zn and Fe) by atomic-absorption spectrometry (AAS) after digestion of both coprolite samples from GS and GM with HNO 3 :HClO 4 by the method 113 . Diacid mixture digested samples were analyzed for transition metal elements using an atomic absorption spectrophotometer (Electronic Corporation of India).
Statistical analysis. Analysis of variance (ANOVA) was used to compare the results between the control and treatment groups followed by post hoc analysis. A significance level of P < 0.05 was used to determine significance in the treatment means using R-software. Regression analysis was carried out using an equation (y = b 2 x 2 − b 1 x + a) and to workout maximum responses in different study parameters were determined from the formula (x = −b 1 /2b 2 ) . In the experiment, the mean differentiation of chemical parameters of nitrogen dynamics was done using student's t-test. Parameters such as pH, moisture content (MC), total organic carbon (TOC), total phosphorus (TP), nitrates, total nitrogen (TN), potassium (K), iron (Fe), zinc (Zn), and the carbon and nitrogen ratio (CN ratio) were utilized to determine correlation matrices affecting coprolites' quality and stability. The findings were plotted and tabulated using principal component analysis.

Conclusion
The study confirms that GM coprolites contains more nitrogen (NH 4 + -N and NO 3 --N) than the GS, which is readily logical when the selective feeding habits of earthworms are considered. At low temperature, simultaneous heterotrophic nitrification and aerobic denitrification (SHNAD) was found to be stable processes and main N transformation mechanism in both substrates. A. caliginous promotes microorganism growth, which would otherwise be severely limited due to harsh winter and low ambient temperature. The study highlights the importance of the SHNAD as a pilot scale process showing positive interaction with A. caliginous contributing in physico-chemical parameters of its coprolites and thus has substantial potential for N removal from wastes at low temperatures. Interaction between psychrophillic earthworms and microbial genera need to be further www.nature.com/scientificreports/ investigated to provide insight evidences of co-occurrence pattern of both, which could help to minimize NH 3 emission by effectively reducing N 2 O.

Data availability
The data sets generated are available as supplementary file.